Lymphocytes bearing high levels of FasL are known to suppress cell-mediated immunity and destroy the efficacy of DNA vaccines. Their ability to kill the antigen-presenting cells can be eliminated by several different mechanisms and we are exploring a number of these mechanisms to find one optimal for promoting cell- mediated responses to SIVGag vaccination. It is our hypothesis that treatment with small molecules that block Fas/FasL signaling could prevent the destruction of antigen-presenting-cells (APC), and allow the development of vigorous anti-retroviral immunity. High levels of CD4+FasL+ cells during AIDS also reduce immunity against opportunistic infections and neoplastic events. Therefore, treatments to block signals delivered by these cells could boost resistance to opportunistic infections and to AIDS-associated cancers. The experimental plan is to develop novel small-molecule inhibitors of Fas/FasL signaling to prevent APC lysis after vaccination. We identified three different strategies for Fas/FasL inhibition which will be compared in mouse immunization studies to identify the strategy, or combination of strategies, most suitable for promoting cell-mediated immunity to a common SIV vaccine antigen, p27Gag. Among our approaches we are investigating the use of pre-ligand assembly domains (PLAD) that are critical for function in TNF receptor superfamily proteins. PLAD represent a novel class of TNF and Fas receptor inhibitors with important advantages for use during DNA vaccination and prime-boost strategies. We propose structural biology studies to address key questions about PLAD structural/functional elements critical for inhibiting Fas/FasL. Our studies will compare inhibitors of FasL signaling that should have short-term effects and highly-specific targets, yet should not interfere with the development of strong functional responses to vaccine antigens PUBLIC HEALTH RELEVANCE: People with AIDS carry a subset of white blood cells that destroy their ability to fight opportunistic infections and to prevent cancer. Attempts to fix this problem with long-lasting solutions would endanger the normal balance of blood cells. Our studies propose to test inhibitors of cell-death signals that would have short-term effects and highly-specific targets, yet would not interfere with the development of strong responses against cancers or infections.